Survey of viral haemorrhagic septicaemia virus in wild fishes in the southeastern Black Sea.

Species diversity in the Black Sea ecosystem has been declining rapidly over the last 2 decades. To assess the occurrence and distribution of viral haemorrhagic septicaemia virus (VHSV) in various wild fish species, a wild marine fish survey was carried out in 2009, 2010, and 2011. The pooled or individual samples of kidney, liver, and spleen of 5025 specimens, belonging to 17 fish species, were examined virologically using cell culture. The cells showing cytopathic effects (CPE) were subjected to ELISA and multiplex reverse transcriptase polymerase chain reaction (RT-mPCR), for VHSV and infectious pancreatic necrosis virus (IPNV), after blind passaging to determine the virus species causing CPE. The virus species and possibility of co-infection with IPNV were verified by the RT-mPCR developed in this study. Twelve species of fish (pontic shad Alosa immaculata, red mullet Mullus barbatus, three-bearded rockling Gaidropsarus vulgaris, black scorpionfish Scorpaena porcus, Mediterranean horse mackerel Trachurus mediterraneus, whiting Merlangius merlangus euxinus, stargazer Uranoscopus scaber, pilchard Sardina pilchardus, garfish Belone belone, round goby Neogobius melanostomus, thornback ray Raja clavata, and anchovy Engraulis encrasicolus) tested positive for VHSV Genotype Ie (VHSV-Ie). Except whiting, pilchard, and round goby, the rest are new host records for VHSV. The extent and spread of VHSV-Ie was significantly higher among bottom fish than among pelagic fish. Sensitivity and specificity of the RT-mPCR developed was sufficiently high, suggesting that this assay may be used for both diagnostic and surveillance testing. According to the RT-mPCR results, IPNV was not present in wild fish. These results support the hypothesis that the VHSV-Ie genotype, highly prevalent among fish species in the Black Sea, may have a serious impact on the population dynamics of wild fish stocks.

Hexamitiasis leads to lower metabolic rates in rainbow trout Oncorhynchus mykiss (Walbaum) juveniles.

This study assessed the effects of Hexamita salmonis (Moore) on metabolism of rainbow trout Oncorhynchus mykiss (Walbaum) and its effect on the host's susceptibility to infectious pancreatic necrosis virus (IPNV) after antiparasitic treatment. Rainbow trout naturally infected with H. salmonis were treated with 10 mg metronidazole kg fish(-1) per day, and their physiological recovery was assessed through measuring resting metabolism on the 7th, 14th, 21st and 28th day after treatment. In addition, we exposed the naïve fish to H. salmonis and measured the resting metabolism (oxygen consumption as mg O2 kg(-1) per hour) on the 10th, 20th and 30th day after the exposure to assess the variation in metabolic rates after infection. Significantly lower rates of metabolic activity (P < 0.05) were anticipated 20 days after infection with H. salmonis compared with the fish infected with H. salmonis for 10 days or with the parasite-free fish. Similarly, the treated fish needed about 20 days to fully recover from hexamitiasis. The susceptibility of rainbow trout to IPNV remained unchanged in the presence of H. salmonis. Weight loss was significantly higher (P < 0.05) in infected than that in the parasite-free fish. Fish should be examined regularly for H. salmonis and treated immediately whether found to prevent economic losses and excessive size variation.

Viral surveillance of cultured Rainbow Trout in the eastern Black Sea, Turkey.

To study the presence and spread of viral fish pathogens in Rainbow Trout Oncorhynchus mykiss in the eastern Black Sea region of Turkey, 172 pooled samples (seven fish per pool) of Rainbow Trout fry from 28 hatcheries were examined from December 2006 to July 2007. Forty-three pools (seven broodfish per pool) of seminal and ovarian fluids from 182 female and 119 male brood Rainbow Trout were also sampled during spawning. Moreover, reproductive fluids (22 pools) of wild trout (Salmo trutta labrax, S. t. caspius, S. t. abanticus, and S. t. macrostigma), captured by electroshocking in the rivers in and around the region, were sampled. Triplicate groups of 40 or 80 Rainbow Trout fry was also challenged with two similar isolates to determine their virulence on trout fry. Enzyme-linked immunosorbent assay was performed on the samples producing cytopathic effect on CHSE-214 cells. The positive results were confirmed with a reverse transcriptase polymerase chain reaction assay. Neither infectious hematopoietic necrosis virus (IHNV) nor viral hemorrhagic septicemia virus (VHSV) was detected during the survey. Of the 28 hatcheries sampled in the Black Sea region, 15 from six provinces tested positive for infectious pancreatic necrosis virus (IPNV) in trout fry. Unexpectedly, all reproductive fluids from both male and female cultured and wild broodstock tested negative for IPNV. Nucleotide sequences of the VP2/NS region of IPNV showed that all isolates collected (n = 38) in the region and surrounding areas belonged to the genogroup III. The findings strongly suggest that IPNV is endemic in the fry of farmed Rainbow Trout within the region. Virus prevention measures should be taken to prevent in-farm spread of these highly contagious, low-virulence isolates.

Monthly occurrence and prevalence of viral haemorrhagic septicaemia virus (VHSV) in whiting Merlangius merlangus.

A field survey was carried out to determine the occurrence and spread of viral haemorrhagic septicaemia virus (VHSV) in whiting Merlangius merlangus (L.), a key prey species in the southeastern Black Sea region. In March, April, September and December 2007 and monthly in 2008, 70 to 105 M. merlangus captured by gill nets were examined in pools of 7 fish per pool (total number of fish = 1603). After determining the seasonal occurrence of VHSV in whiting, 43, 80 and 88 whiting were sampled individually in January, February and March 2009, respectively, to determine age and size of whiting commonly infected with VHSV. The samples were tested in duplicate using BF-2 and EPC cell lines for virus isolation. Positive cultures (those demonstrating cytopathic effect, [CPE]), were tested further with a commercial ELISA kit to confirm that the observed CPE was caused by VHSV. VHSV was detected in M. merlangus only between February and April following a drop in the surface water temperature to 9 degrees C. All infected fish from the sampled whiting were asymptomatic carriers with low levels of virus. The estimated prevalence from pooled samples ranged between 1.35% (2.5 to 97.5 percentiles; 0.04 to 7.32%) to 3.14% (0.36 to 10.95%), whereas prevalence estimated from individual sampling was 7, 5 and 1% in January, February and March, respectively. Individual sampling also revealed that prevalence was higher among young fish (1 to 1.5 yr old) and decreased with increasing age. This is the first report of VHSV infection in whiting in the Black Sea.

A stochastic modelling approach to describing the dynamics of an experimental furunculosis epidemic in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

A susceptible-infected-removed (SIR) stochastic model was compared to a susceptible-latent-infectious-removed (SLIR) stochastic model in terms of describing and capturing the variation observed in replicated experimental furunculosis epidemics, caused by Aeromonas salmonicida. The epidemics had been created by releasing a single infectious fish into a group of susceptible fish (n = 43) and progress of the epidemic was observed for 10 days. This process was replicated in 70 independent groups. The two stochastic models were run 5000 times and after every run and every 100 runs, daily mean values of each compartment were compared to the observed data. Both models, the SIR model (R(2) = 0.91), and the SLIR model (R(2) = 0.90) were successful in predicting the number of fish in each category at each time point in the experimental data. Moreover, between-replicate variability in the stochastic model output was similar to between-replicate variability in the experimental data. Generally, there was little change in the goodness of fit (R(2)) after 200 runs in the SIR model whereas 500 runs were necessary to have stable predictions with the SLIR model. In the SIR model, on an individual replicate basis, approximately 80% of 5000 simulated replicates had R(2) = 0.7 and above, whereas this ratio was slightly higher (82%) with the SLIR model. In brief, both models were equally effective in predicting the observed data and its variance but the SLIR model was advantageous because it differentiated the latent, i.e. infected but not having the ability to discharge pathogen, from the infectious fish.

Effects of host density on furunculosis epidemics determined by the simple SIR model.

To determine the transmission dynamics of furunculosis, Chinook salmon, Oncorhynchus tshawytscha held at various densities were challenged with a single infectious fish. Data from the in vivo experiments were compared to predicted values from a susceptible--infected--removed (SIR) model. Seven different densities were tested including 9.13, 4.56, 0.72, 0.36, 0.19, 0.06 and 0.03 fish/L and each produced different transmission coefficients (beta) of 0.01, 0.019, 0.0051, 0.0076, 0.0001, 0.0005 and 0.00, respectively. Furunculosis related mortality rates decreased as density of the host decreased. Mortality rates at the highest fish densities where disease specific mortality (DSM) was observed were 0.42, 0.44, 0.18 and 0.02 infected animals per day. The natural mortality rate also followed a similar pattern with 0.005, 0.005, 0.001, 0.005, 0.0001, 0.000 and 0.000 surviving animals per day. The results indicated that the transmission coefficient and the DSM of furunculosis was dependent on host density. It is possible that fish behavioral changes at the lower densities caused the reproductive rate of disease to be persistent even at very low densities. In summary, the simple SIR model suggested that low mortality does not always mean low prevalence of furunculosis in a given population. This is the first study evaluating host density as a risk factor for fish diseases using experimental furunculosis data.

Evaluation of an experimental Aeromonas salmonicida epidemic in chinook salmon, Oncorhynchus tshawytscha (Walbaum).

To determine the dynamics of the transmission of Aeromonas salmonicida ssp. salmonicida infection, chinook salmon, Oncorhynchus tshawytscha, were exposed to bacteria by cohabitation. The latent period (time between exposure and infectivity) was determined by exposing a group of chinook salmonid fingerlings to A. salmonicida by bath, then, at daily intervals, by holding five exposed (donor) fish with approximately 50 naive fish for 24 h. The latent period was 3 days post-infection and the time period between the initial exposure to bacteria and the beginning of bacterial shedding was 4.5 days for the same animals. The prevalence and intensity of infection in the donor fish, to which recipient fish were exposed, i.e. the level of exposure, was highly correlated with the development of disease in recipient (susceptible) chinook salmon (r2 = 0.57). An experiment was conducted to determine the daily progress of infection and development of a furunculosis epidemic among recipient fish by cohabiting a single exposed fish with 43 unexposed salmon. At daily intervals, all fish (in seven treatment tanks and one control tank daily) were sacrificed and tested for the presence of A. salmonicida in the kidney (n = 3520). Over 10 days, mean prevalence among recipient fish reached 75% and disease related mortality exceeded 50%. Bacterial concentrations in the water continued to increase over the duration of the experiment in concert with the number of infected animals present in the population.

A deterministic model for the dynamics of furunculosis in chinook salmon Oncorhynchus tshawytscha.

Studies were undertaken to determine the parameters of transmission of Aeromonas salmonicida in chinook salmon Oncorhynchus tshawytscha, and to develop a deterministic model of the dynamics of experimental furunculosis. For determination of disease transmission coefficient (beta), disease-related mortality rate (alpha) and natural mortality rate (gamma), fish in 70 tanks (approximately 42 fish tank(-1)) were each exposed to a single infectious donor fish, 7 tanks were randomly selected daily and all individuals were examined for the presence of A. salmonicida in the kidney. The proportion of susceptible (S), infected (I) and removed (R, dead) individuals were determined daily. The parameters beta, alpha, gamma, reproductive ratio (R0) and threshold density were estimated to be 0.0214 infected ind. d(-1), 0.29 infected ind. d(-1), 0.00015 ind. d(-1), 3.23 and 13.56 ind., respectively. Using these parameters, a deterministic disease model of A. salmonicida infection as a cause of furunculosis was constructed. The net rate at which new individuals became infected (the incidence rate) per unit time was proportional to S x I x beta. The model-produced data for S were significantly associated with experimental data (r2 = 0.92). In brief, a simple SIR (susceptible-infected-removed) model was successfully utilized to simulate observed data

Primary lymphoma of bones.

Primary malignant lymphoma of bone (PLB) is an uncommon tumour. A survey of 278 primary malignant cases in our clinic showed that 20 cases of PLB were registered between 1986 and 1997. Fourteen of the 20 cases underwent surgical excision. The mean follow-up time was 36.3 months. The rate of response to treatment was 65% with a rate of complete recovery of 55%. The clinical stage of tumours correlated well with the prognosis. Our results seem worse than most of the series in the literature. A high proportion of stage IV disease and pathological fractures at presentation may be a factor.

Giant cell tumor of bone.

Seventy-two cases of giant cell tumor (GCT) of bone were diagnosed in our clinic between 1986 and 1998. The mean age of the patients was 35 (range: 11 to 63 years) and the female to male ratio was 1.3:1. Fifty-four percent of the cases had involvement about the knee (distal femur, upper tibia, and upper fibula). Four patients had a malignant form of the disease and another four had the disease before closure of the physis. There was no case of multicentric involvement. Nine patients presented with pathological fracture. Forty-seven patients had histological grading done. Twenty three (49%) had grade I, 17 (36%) had grade II, and seven (15%) had grade III disease. The mean follow-up time was 59.6 months (range: 24 to 142 months) for patients at least two years follow-up. There was a 33% complication rate.